Cellular lipid homeostasis is required to maintain bilayer fluidity, membrane impermeability, and organelle identity. Disturbances in systemic lipid homeostasis lie at the core of the pathologies for both coronary artery disease and obesity related type II diabetes. The long-term goal of this research is to translate knowledge of cellular regulatory events to that of the whole organism and to advance our understanding of these increasingly widespread diseases. As a first step toward this goal, we will use cholesterol as a model lipid to understand how cells measure levels of these largely insoluble molecules and in turn modulate their production. Cholesterol homeostasis in mammalian cells is regulated by a feedback mechanism that monitors the level of cholesterol in membranes and alters transcription of genes required for cholesterol supply. Transcription of these genes is controlled by the ER membrane-bound transcription factor called SREBP that is activated and released from the membrane by proteolysis in sterol-depleted cells. To accelerate discovery of sterol homeostasis regulators, we are studying the SREBP pathway in the fission yeast Schizosaccharomyces pombe. Yeast SREBP, called Sre1, functions in a new oxygen sensing pathway that mediates adaptation of cells to low oxygen. Interestingly, sterols regulate Sre1 activity through a novel mechanism in fission yeast, and evidence also indicates that Sre1 cleavage is mediated by a unique proteolytic system. In this project, a combination of genetic, molecular, and biochemical approaches will be used to accomplish the following specific aims: 1) To identify genes required for Sre1 cleavage using a genetic selection; 2) To define the machinery for Sre1 cleavage; and 3) To define the mechanism of sterol-regulated Sre1 cleavage. The long-term goal of this project is to use S. pombe as a genetic model to understand how cells measure levels of insoluble, membrane-embedded cholesterol. The expectation is that these studies will describe new mechanisms for lipid sensing and proteolysis that will advance our understanding of the mammalia SREBP pathway and eukaryotic cell biology.